1. Field of the Invention
The present invention relates to gene regulation therapy involving ferritin. More specifically, the invention relates to the use of Ferritin-H and derivative proteins thereof for regulation of genes related to iron metabolism and regulation.
2. Prior Art
Background for Sickle Cell Disease
Hematopoiesis, or the formation of blood cells, begins in the developing human embryo as clusters of stem cells called blood islands. These cells appear in the yolk sac at about the third week of development and, at about the third month, migrate to the developing liver which becomes the principal site of blood cell formation. Although the spleen, lymph nodes and bone marrow all make small contributions to blood cell development, not until the fourth month does the bone marrow become the principal site of hematopoiesis. At birth, virtually all blood cells originate from the bone marrow. Although small foci of blood-forming cells sometimes persist in the liver for longer periods of time, hepatic blood cell formation has decreased to a trickle. At this time, all of the marrow is actively forming blood cells and continues to do so until after puberty when, at about 18 years of age, the principal sites of blood cell formation become the marrow of the vertebrae, ribs, sternum, skull, pelvis and the proximal epiphyseal regions of the femur and humerus. These areas represent only about half of the available marrow. The cavities which remain are filled with yellow-fatty tissues.
In the adult, hematopoiesis involves the bone marrow, the lymph nodes and the spleen. These organs and associated tissues are traditionally divided into myeloid and lymphoid tissue-types. Myeloid tissues and the cells derived from the myeloid tissue include the erythrocytes, platelets, granulocytes and monocytes. Lymphoid and lymphoid-derived tissues include the thymus, lymph nodes and spleen. The myeloid/lymphoid division is somewhat artificial as these two types of tissues are believed to originate from a single pluripotent stem cell.
Lymphoid and myeloid stem cells, formed from division of the pluripotent cell, are precursors for all subsequent cell types. The committed cell-types for the lymphoid stem cell include the pro-T cells which form mature T cells and the pro-B cells which differentiate into plasma cells. Intermediate cell types can be distinguished based on cell-surface phenomenon such as the expression of immunoglobulin heavy and light chain, Ia protein and other cell surface markers. The three committed cell-types for the myeloid stem cell include E/mega cells which differentiate into the erythrocyte-burst forming unit (BFU-E) followed by the erythrocyte-colony forming unit cells (CFU-E) and megakaryocyte-CFU cells (CFU-mega), granulocyte/macrophage-CFU cells (CFU-G/M) which differentiate into CFU-G and CFU-M cells, and the eosinophil-CFU cells (CFU-Eo) which ultimately form mature eosinophils. Although these committed cell types reside mainly in the marrow, some circulate throughout the body in the blood stream.
The relative proportions of cell types in the bone marrow have a myeloid/erythroid ratio of about three to one comprising about 60% granulocytes and their precursors, about 10% lymphocytes and their precursors, about 20% erythrocytes and their precursors, and about 10% unidentified cells. The predominant myeloid cell types in the marrow cavity are the myelocytes, metamyelocytes and granulocytes. The predominant cell types in the erythroid compartment are the polychromatophilic and orhtochromic normoblasts. Under conditions of normal iron metabolism, about 30% to 40% of the normoblasts contain scattered ferritin granules. These cells are referred to as sideroblasts and the iron granules they contain are reservoirs drawn from as the cells insert iron into protoporphyrin to form heme. The production of heme and the production of globin are precisely balanced within the cell. If either is hindered or depressed, for whatever reason, excess ferritin accumulates in the sideroblasts. This increased iron accumulation can be visualized in the mitochondria, the loci of heme synthesis.
The major function of red blood cells is to transport oxygen to tissues of the body. Minor functions include the transportation of nutrients, intercellular messages and cytokines, and the absorption of cellular metabolites. Anemia, or a loss of red blood cells or red blood cell capacity, can be grossly defined as a reduction in the ability of blood to transport oxygen and may be acute or chronic. Chronic blood loss may be caused by extrinsic red blood cell abnormalities, intrinsic abnormalities or impaired production of red blood cells. Extrinsic or extra-corpuscular abnormalities include antibody-mediated disorders such as transfusion reactions and erythroblastosis, mechanical trauma to red cells such as micro-angiopathic hemolytic anemias, thrombotic thrombocytopenic purpura and disseminated intravascular coagulation. In addition, infections by parasites such as Plasmodium, chemical injuries from, for example, lead poisoning, and sequestration in the mononuclear system such as by hypersplenism can provoke red blood cell disorders.
Hemoglobin comprises four protein chains, two alpha chains and two beta chains (α2 β2), interwoven together, each with its own molecule of iron and with a combined molecular weight of about 68 kD. The hemoglobin macromolecule is normally glycosylated and upon absorbing oxygen from the lungs transforms into oxyhemoglobin (HbO2). There are at least six distinct forms of hemoglobin, each expressed at various times during development. Hemoglobin in the embryo is found in at least three forms, Hb-Gower 1 (ξ2 ε2), Hb-Gower 2 (α2 ε2) and Hb-Portand (ξ2 γ2). Hemoglobin in the fetus comprises nearly totally HbF (α2 γ2), whereas hemoglobin in the adult contains about 96% HbA (α2 β2), about 3% HbA2 (α2 δ2) and about 1% fetal HbF (α2 γ2). The embryonic switch of globin expression from ξ- to α- and from ε- to γ- begins in the yolk sac. However, chains of embryonic ξ- and ε- have been found in the fetal liver and complete transition to the fetal form does not occur until late in fetal development. The fetal switch from γ- to β- begins later in erythropoiesis with the amount of β-globin produced increasing throughout gestation. At birth, β-globin accounts for about 40% of non-α-globin chain synthesis and thereafter continues to rapidly increase.
Defects or mutations in globin chain expression are common. Some of these genetic mutations pose no adverse or only minor consequences to the person, however, most mutations prevent the formation of an intact or normal hemoglobin molecule through a functional or structural inability to effectively bind iron, an inability of the chains or chain pairs to effectively or properly interact, an inability of the molecule to absorb or release oxygen, a failure to express sufficient quantities of one or more globin chains or a combination of these malfunctions. For example, substitutions of valine for glutamic acid at the sixth position of the β chain produces HbS and was found to occur in about 30% of black Americans. In the HbS heterozygote, only about 40% of total hemoglobin is HbS with the remainder being the more normal HbA.
Upon deoxygenation, HbS molecules undergo aggregation and polymerization ultimately leading to a morphological distortion of the red cells which acquire a sickle or holly-leaf shape. Sickling has two major consequences, a chronic hemolytic anemia and an occlusion of small blood vessels that results in ischemic damage to tissues. Further, when exposed to low oxygen tensions, polymerization converts HbS hemoglobin from a free-flowing liquid to a viscous gel. Consequently, the degree of pathology associated with sickle cell anemia can be correlated with the relative amount of HbS in the patient's system.
Individuals with severe sickle cell anemia develop no symptoms until about five to six months after birth. In these infants it was determined that fetal hemoglobin did not interact with HbS and, as long as sufficient quantities were present, could modulate the effects of HbS disease. This modulating effect of γ-globin is also observed with other β-globin disorders, such as HbC and HbD, and other mutations of the β chain. HbS polymerization is also significantly affected by the hemoglobin concentration of the cell. The higher the HbS concentration, the greater the chances for contact between two or more HbS molecules. Dehydration increases hemoglobin concentration and greatly facilitates sickling.
To some extent, sickling is a reversible phenomenon. With increased oxygen tensions, sickled cells depolymerize. This process of polymerization-depolymerization is very damaging to red cell membranes and eventually leads to irreversibly sickled cells (ISC) which retain their abnormal shape even when fully oxygenated. The average ISC survives for about 20 days in the body, as compared to the normal 120 day life span. Individuals with HbS syndromes have frequent infections, chronic hemolysis with a striking reticulocytosis and hyperbilirubinemia. The course of the disease is typically punctuated with a variety of painful crises called vaso-occlusive crises. These crises represent episodes of hypoxic injury and infarction in the organs, abdomen, chest, extremities or joints. Leg ulcers are an additional manifestation of the vaso-occlusive tendency of this disease. Central nervous system involvement is common producing seizures and even strokes. Aplastic crises, also common, represent a temporary cessation of bone marrow activity and may be triggered by infections, folic acid deficiency or both. Crises are episodic and reversible, but may be fatal. Damage from crisis episodes tends to be cumulative and even in those individuals with milder forms of sickle cell disorders, life-spans can be greatly reduced. Absent alternative intervention, patients typically die before the age of 30.
Anti-gelling compounds including clofibric acid (ClC6 H5 OC(CH3)2COOH), p-chlorophenoxy acetic acid (ClC6H5OCH2COOH), and phenoxy acetic acid (C6 H5 OCH2 COOH) have been shown to prophylactically inhibit polymerization in artificially deoxygenated blood. It was speculated that these compounds may be useful in a narrow respect to prevent blood cell sickling in sickle cell disease. Such treatments may potentially decrease the frequency of symptomatic episodes caused by vaso-occlusive crises if enough of the chemical can be administered to bind all hemoglobin in the body.
The thalassemia syndromes are a heterogenous group of disorders all characterized by a lack of or a decreased synthesis of the globin chains of HbA. Deficiencies of β-globin expression are referred to as β-thalassemias and deficiencies of α-globin, α-thalassemias. The hemolytic consequences of deficient globin chain synthesis result from decreased synthesis of one chain and also an excess of the complementary chain. Free chains tend to aggregate into insoluble inclusions within erythrocytes causing premature destruction of maturing erythrocytes and their precursors, ineffective erythropoiesis, and the hemolysis of mature red blood cells. The underlying defects of hemoglobin synthesis have been elucidated over the years and largely reside in the nucleic acid sequences which express or control the expression of α- or β-globin protein.
Mammalian globin gene expression is highly regulated during development. The basic structure of the α- and β-globin genes are similar as are the basic steps in synthesis of α- and β-globin. There are at least five human α-globin genes located on chromosome 16 including two adult α-globin genes of 141 amino acids that encode identical polypeptides and differ only in their 3′-untranslated regions, one embryonic a gene Z(ζ), and at least two pseudo-α genes, psi zeta (ΨZ) and omega alpha (ωα).
The human β-globin gene cluster includes one embryonic gene, epsilon (ε), two adult beta globin genes, beta (β) and delta (δ), two fetal beta globin genes G-gamma (G-γ) and A-gamma (A-γ.), which differ by only one amino acid, and at least one pseudo-beta gene, psi beta (Ψβ). All are expressed from a single 43 kilobase segment of human chromosome 11. Fetal β-type globin, or γ-globin, is expressed in the earliest stages of mammalian development and persists until about 32 to 34 weeks of gestation. At this stage, the adult forms of β-globin begin to be expressed and substitute for the fetal proteins. Studies correlating clinical hematological results with the locations of various mutations that correspond to switching indicate that a region located upstream of the 5′-end of the δ-gene may be involved in the cis suppression of γ-gene expression in adults. The stimulus for this switch from fetal to adult protein is unknown.
Each β-globin gene comprises three exons which encode about 146 amino acids, two introns and a 5′-untranslated region containing the promoter sequences and a 3′ untranslated region. Biosynthesis of β-globin begins with transcription of the entire gene followed with RNA processing of the message, removal of the introns by splicing, poly A addition, capping and post-transcriptional modifications. The mature mRNA molecule is exported from the nucleus and translated into β-globin. Defects in each of these functions have been found associated with specific thalassemias. Identified mutations include single-nucleotide deletions, insertions and substitutions, frame shift mutations, deletions of entire segments of coding or controlling regions, improper termination signals, aberrant splicing signals, and multiple mutations. β°-thalassemias are characterized by a complete absence of any β-globin chains; β+-thalassemias are characterized by a detectable presence of a reduced amount of β chains.
There are three principal categories of β-thalassemia, thalassemia major, thalassemia intermedia and thalassemia minor. Patients with thalassemia minor may be totally asymptomatic and are genotypically β+/β or β°/β. Although red cell abnormalities can be detected, symptoms are mild. Thalassemia intermedia patients are most often genotypically β+/β+ or β°/β and present severe symptoms which can be alleviated with infrequent blood transfusions. In contrast, thalassemia major patients are genotypically β°/β°, β°/β+ or β+/β+, and require regular and frequent transfusions. Children suffer from severe growth retardation and die at an early age from the profound effects of anemia. Those that survive longer suffer from morphological changes. The face becomes distorted due to expansion of marrow within the bones of the skull, hepatosplenomegaly ensues, there is a delayed development of the endocrine organs including the sexual organs, and a progressive iron overload with secondary hemochromatosis.
There are two direct consequences of β-thalassemia. First, there is an inadequate formation of HbA and, therefore, an impaired ability to transport oxygen. There are also multiple effects attributable to an imbalance between α- and β-chain synthesis. Surprisingly, the pathological consequences of globin chain imbalance appears to be the more severe. Free α chains form unstable aggregates that precipitate within red cell precursors in the form of insoluble inclusions. These inclusions damage cellular membranes resulting in a loss of potassium. The cumulative effect of these inclusions on the red blood cells is an ineffective erythropoiesis. An estimated 70% to 85% of normoblasts in the marrow are eventually destroyed. Those that do escape immediate destruction are at increased risk of elimination by the spleen where macrophages remove abnormal cells. Further, hemolysis triggers an increased expression of erythropoietin which expands populations of erythroid precursors within bone marrow and leads to skeletal abnormalities. Another severe complication of β-thalassemia is that patients tend to have an increased ability to absorb dietary iron. As most treatments for thalassemia involve multiple transfusions of red blood cells, patients often have a severe state of iron overload damaging all of the organs and particularly the liver. To reduce the amount of iron in their systems, iron chelators are typically administered. Although helpful, patients succumb at an average of between about 17 to 35 years of age to the cumulative effects of the disease and iron overload.
Genotypic variations in healthy individuals have been identified wherein adult β-globin is not formed, but severe complications are avoided. These patients constituitively express fetal or γ-globin protein in amounts sufficient to substitute for the missing β-globin protein. This hereditary persistence of fetal hemoglobin (HPFH) may involve one or both of the fetal β-globin genes, A-γ and G-γ. Apparently, consistent production of either γ-globin protein accomplishes the necessary functions of the abnormal or missing β-globin protein.
A variety of small molecules have been shown to effect hemoglobin or fetal globin expression. Early experiments demonstrated that acetate (CH3COOH), propionate (CH3CH2COOH), butyrate (CH3CH2CH2COOH) and isobutyrate (CH3CH(CH3)COOH) all induced hemoglobin synthesis in cultured Friend leukemia cells. Additional studies showed that polar compounds, such as acid amides, and fatty acids could stimulate the expression of both fetal and adult globin genes in murine erythroleukemia cells. Hydroxyurea (H2NCONHOH), another relatively small molecule, was found to stimulate globin expression. Stimulation, however, does not appear to be very specific to fetal globin. Hydroxyurea is currently the only drug used to treat sickle cell disease. However, there is great concern that an antineoplastic ribonucleotide reductase inhibitor is be carcinogenic, its carcinogenic properties make its widespread and long term use as a pharmaceutical a questionable practice. There is a strong need to find methods of treating sickle cell disease that do not include the patient's exposure to other risks.
Expression from the γ-globin genes has been successfully manipulated in vivo and in vitro using agents such as cytosine arabinoside (AraC), a cytotoxic agent that induces fetal reticulocyte production, and 5-azacytidine (AZA), a well-known DNA methylase inhibitor. Continuous intravenous administration of AZA produced a five- to seven-fold increase in γ-globin mRNA of bone marrow cells. Additional studies have shown that there are significant alterations in the population of stem cells in the bone marrow after AZA treatment. These experiments indicate that AZA's effects may be more attributable to reprogramming and recruitment of erythroid progenitor cells than to any direct effects on specific gene expression. Many of these agents including AZA, AraC and hydroxyurea are myelotoxic, carcinogenic or teratogenic making long-term use impractical.
One of the major breakthroughs in the treatment of hemoglobinopathies was made when it was discovered that butyric acid (butanoic acid; CH3CH2CH2COOH) accurately and specifically stimulated transcription of the human fetal (γ) globin gene. These findings were quickly confirmed in vivo wherein it was shown that pharmacological doses of butyric acid greatly increased expression of fetal globin in adult chickens rendered anemic by injections with phenylhydrazine. It was speculated that histone acetylation, a known effect of butyric acid, may be at least partly responsible for increased fetal gene expression.
Over 50 derivatives of butyric acid have since been found to be effective in stimulating fetal globin production. Some of these include butyric acid salts such as sodium and arginine butyrate, α-amino-n-butyric acid (butyramide; CH3CH2CH2CONH2), and isobutyramide (CH3CH(CH3)CONH2). Although promising in pilot clinical studies, treated patients were unable to maintain adequate levels of fetal globin in their system. It was later determined that many of these forms of butyric acid had extremely short-half lives. Oxidation in the serum, clearance by hepatocytes and filtration through the kidneys rapidly eliminated these agents from the patient's system. With others, patients rapidly developed tolerance or metabolites of compounds had the opposite of the desired effect.
Recently, a number of aliphatic carboxylic acids were tested for their ability to specifically increase fetal globin expression in K562 human erythroleukemia cells. Although longer chains were considered toxic to cells, propionate (CH3CH2 COOH) and valerate (pentanoic acid; CH3CH2CH2CH2COOH) were found to be most effective. Butyrate (CH3(CH2)2COOH), caproate (CH3(CH2)4COOH), caprylate (CH3(CH2)6COOH), nonanoate (CH3(CH2)7COOH), and caprate (CH3(CH2)6COOH) produced much less of an effect. Phenyl acetate (C6H5CH2COOH) and its precursor, 4-phenyl butyrate (C6H5CH2CH2CH2COOH), were found to decrease fetal globin expressing reticulocyte proliferation, but increase relative proportions of fetal globin per cell in cultured erythroid progenitor cells. Acetate (CH3COOH), a metabolic product of butyrate catabolism, increased both erythrocyte precursor populations and also fetal globin synthesis. However, these studies also demonstrated that positive effects could only be maintained for very short periods of time.
Other methodologies to increase fetal globin expression have focused on recruitment and reprogramming of erythroid progenitor cells to express fetal globin. Agents tested in vivo or in vitro using this approach include hematopoietic growth factors such as erythropoietin (EPO), granulocyte/macrophage-colony stimulating factor (GM-CSF), and interleukin-3 (IL3). Each of these factors were found to increase fetal globin synthesis in tissue culture cells.
Other agents shown to affect fetal globin expression include activin and inhibin. Inhibin, a disulfide linked hormone of two subunits, suppresses secretion of follicle-stimulating hormone from the pituitary gland. Activin, sometimes referred to as erythroid differentiating factor (EDF) or follicle-stimulating hormone releasing protein (FRP), is also a hormone and both of these macromolecules induced hemoglobin accumulation in cultured human erythrocytes (S. P. Perrine et al., Blood 74:114a, 1989). Recently, studies have shown that steel factor, a product of the mouse steel locus, is also capable of influencing fetal globin synthesis in erythroid progenitors.
Several studies have focused on the mechanism whereby butyric acid and other small organic molecules have been able to stimulate fetal globin expression. Experiments with cells in culture have indicated that butyric acid may act by increasing the level of histone acetylation by, possibly, decreasing the activity of one or more histone deacetylase. Resulting histone hyperacetylation may produce nucleosome unfolding and thereby increased gene expression. Other studies have indicated that hypo-methylation of the area of DNA around the β gene complex correlates with increased γ-globin gene expression in thalassemic patients. Alternatively, butyric acid and other small molecules may function to increase specific gene expression by acting directly on agents which regulate transcription, the so-called transcription factors. These factors bind to sequence-specific sites along the genome at areas which control the expression of proximally located genes.
In contrast to the human a-globin gene locus, the β-locus has been analyzed in great detail due, in part, to the identification of multiple mutations of β-globin genes in HPFH patients. The β-locus contains a large upstream sequence referred to as the locus control region (LCR), extending 8-16 kbp 5′ of the epsilon gene. This sequence is divided into four DNase hypersensitive sites, HSS 1-5, that contain enhancer sequences, silencer sequences, transcription factor binding sites and other cis acting sequences. Each of the genes of the β-globin cluster contains its own promoter which acts in concert with enhancer elements in the LCR. In fact, deletion of the LCR results in a thalassemic syndrome with little to no β-globin expression. These results indicate that the β-globin gene expressed may exert a competitive interaction over the LCR so that its enhancer effect is only available to a single gene at any given time of development.
A number of transcription factors have been identified in the β-locus which are thought to alter the level of β-globin gene expression. An enhancer element of the LCR has been shown to contain a pair of binding sites for nuclear factor E2 (NF-E2) which overlaps a tandem set of binding sites for transcription factor AP-1.NF-E2, a hematopoietic-specific basic leucine zipper protein, and AP-1 binding sites have been located on a variety of globin genetic elements. Recently, a conserved sequence (CS) located upstream of the AP-1/NF-E2 site has been proposed to augment enhancing activity.
Additional factors that bind to elements within the promoters of the β-globin cluster have been identified. The CAT box displacement protein (CDP) binds to the sequence CAAT, located about 50 bp upstream of many gene promoters. Another fairly ubiquitous transcription factor, SP1, binds to positions −140 and −202, and possible additional sites as well. TAFII110 has been shown to binds to the TATA box of many of the β-globin promoters. Transcription factor GATA-1, binds to the transcription initiation site (GATA) and may be displaced by TFIID when forming an active initiation complex. Another erythroid-specific factor, YY1, binds to at least 11 sites distributed throughout the globin regulatory region.
Recently, a factor has been identified that may be involved in the developmental regulation of hemoglobin expression. This factor, termed the stage selector protein (SSP), binds to a site located about 50-60 bps upstream of the gamma globin promoter referred to as the stage selector element (SSE). The SSE is also the site where a number of mutations have been found in HPFH syndrome patients. SSP has been purified from K562 cell nuclear extracts and its relatively fetal and erythroid specificity has been attributed to a heterodimeric partner protein of 40-45 kD termed CP2 which selectively allows assembly of the SSP complex on the SSE, and also on sites within the ε promoter, and subsequent interaction with RNA polymerase.
Elucidating the mechanism of developmental hemoglobin (Hb) switching may allow the reactivation of fetal Hb in adult humans with sickle cell disease or β-thalassemia, a manipulation that alleviates the clinical manifestations of these diseases. Inactivation of the mutated form of the adult β-globin gene that causes sickle cell disease is also of clinical value, since it results in a compensatory increase in γ(fetal)-globin expression.
It is clear that developmental regulation of globin genes involves multiple trans-acting factors, and the mechanism of switching is likely to require chromatin remodeling and interactions among protein factors bound at a variety of DNA regions. Although a few of the known DNA-binding proteins display some developmental specificity (e.g., erythroid Kruppel-like factor, EKLF, a positive regulator of the adult β-globin gene;(Donze, D., Townes, T. M. & Bieker, J. J. (1995) J Biol Chem 270,1955-9; and FKLF-2, which activates the γ globin genes, but also, to a lesser degree activates the ε- and β-globin genes—Asano, H., Li, X. S. & Stamatoyannopoulos, G. (2000) Blood 95, 3578-3584.), the precise combination of factors that mediate Hb switching and exactly how they do so are not clear.
Human K562 cells treated with hemin exhibit an Hb phenotype similar to embryonic erythroid cells, expressing primarily e- and g-globins but no adult β-globin, i.e., embryonic red cells of humans and other vertebrates also contain a large amount of ferritin of the specialized-cell (H) type (which stores iron for use by other cells, mainly), whereas erythrocytes of adults contain much less ferritin of the housekeeping type which stores iron for self/intracellular use.
After a long series of experimentation, the inventors discovered that in CV-1 cells, an expression clone of human H-ferritin down-regulates expression of an EKLF-stimulated β-globin promoter-driven CAT reporter gene. The inventors further show that ferritin in nuclear extracts of K562 cells can bind 5′-β-globin DNA between −153 and −148 and that a highly conserved hexanucleotide sequence CAGTGC is required for this binding. This sequence is essential for b-globin expression in DMSO-induced MEL cells (deBoer, E., Antoniou, M., Mignotte, V., Wall, L. & Grosveld, F. (1988) Embo J 7, 4203-4212) and is part of the binding site of a purported b-globin repressor in uninduced MEL cells (Macleod, K. & Plumb, M. (1991) Mol Cell Biol 11, 4324-32). When this CAGTGC motif is mutated, in vitro binding is reduced approximately twenty fold. The ability of ferritin-H to repress in this system is abolished, but EKLF-stimulation is retained, when the −153 /−148 ferritin binding site is mutated in the co-transfected β-globin-reporter plasmid. These results show that ferritin H can repress the human adult β-globin gene by binding the promoter in a sequence-specific manner. The biology of this ferritin-family protein and its binding site, as well as its demonstrated function in transient assays, suggest that in K562 cells it is indeed functioning as a β-globin repressor. As noted above, such a repressor is useful in ameliorating sickle cell and other genetic diseases.
Studies have shown that ferritin-H exhibits the most efficient ferroxidase activity when it is expressed at roughly the same levels as ferritin-L. Equal expression levels result in the highest number of ferritin-H/ferritin-L heteropolymers. The heteropolymeric form of the 24-mer ferritin complex is the most efficient at converting the ferrous ion to the ferric ion and at sequestering iron ions. This suggests that maintaining equal concentrations of ferritin-H and ferritin-L is most likely to result in proper iron management. Increasing levels of ferritin-H would result in the formation of ferritin-H homopolymers. Ferritin-H homopolymers exhibit low ferroxidase activity. It would be expected that this would lead to higher levels of the more harmful ferrous ion and have adverse affects on the cells. However, the inventors have discovered that the gene regulatory functions of ferritin-H causes just the opposite to occur.
Background for skin cancer and other cancers:
Ultraviolet (UV) light is known to be damaging to human skin and has been implicated in the etiology of skin cancers. Recent studies have revealed that ferritin is elevated in cultured skin cells exposed to UV light, and it has been postulated that the increased ferritin represents the skin cell's attempt to protect itself from free radical damage by binding and sequestering iron which could, in turn, cause oxidative and free radical-mediated damage.
The rationale for other cancers is similar. Iron has been implicated as an etiologic or exacerbating agent in skin cancer, hepatomas (liver cancer), renal cell carcinoma (kidney cancer), neuroblastomas, leukemias, and breast cancer. The inventors propose that ferritin-H will be protective against carcinogenic events in cells that give rise to all of these cancers. The inventors' present rationale is that by treating human skin in such a way as to transfect them with a ferritin-H-subfamily peptide or gene that will express the peptide, protection from UV-induced damage can be provided to the cells. Ferritin-H-subfamily peptides are superior in this regard since they can sequester iron and not release it readily and can do so without altering normal aspects of the cells iron metabolism and other functions. Ferritin-L-subfamily peptides, on the other hand, are likely to cause even more harm in that they readily give up iron which would exacerbate the problem by increasing free iron and radical generation. Thus, delivering a ferritin-H-subfamily peptide or a gene (expression clone) for the peptide to the target cells would be protective and/or corrective of events that lead to cancer. Similarly, agents that would activate the endogenous ferritin-H-subfamily gene or genes would be beneficial in the same ways.
It is realized that all human ferritins, even those highly enriched in ferritin-L, require a small amount of ferritin-H and its associated ferrooxidase activity to carry out the functions of iron storage and release. It is the balance between ferritins L and H that is critical. Increasing the balance in favor of ferritin-H, even to the point of great excess of ferritin-H, appears to mediate a cell's return to healthy iron management
Background for neurodegenerative diseases:
The distribution of free iron and of ferritin both change during brain development in animals and humans. Increased iron is found in the basal ganglia, beginning early in the disease process, in both Parkinson's disease and Huntington's disease. There is an increase in iron in several areas of the brain in Alzheimer's disease, in other dementias, and in aging; and the distribution of isoferritins in a variety of brain areas is different and changes in the above diseases. H-ferritin, but not L-ferritin, is present in the nucleus of neuronal cells in the cortex of developing rat brains and may be protective against oxidative damage that would be caused by free iron. Rationale: Ferritin-H decreases in critical brain cells during aging and neurodenerative diseases, whereas free iron and iron released from localized ferritin-L are implicated in oxidative damage in diseases and dementia. Ferritin-H or a related subfamily peptide will be protective against a variety of neurodegenerative changes associated with aging, the above diseases and dementias. Likewise, an expression clone of a ferritin-H-subfamily gene and/or a regulator of ferritin-H-subfamily genes, if delivered to the appropriate brain area and to specific cells, is predicted to be protective.
Background for Friedreich's ataxia and related neuromuscular disorders:
Deletion of YDL120, the yeast homologue of the human gene responsible for Friedreich's ataxia, elicits decreased cellular respiration associated with decreased cytochrome c oxidase activity and, in certain nuclear backgrounds, mitochondrial DNA is lost. In the null mutants, the cellular growth is highly sensitive to oxidants, such as H2O2, iron and copper; and ferrous sulfate elicits loss of mitochondrial DNA. Mitochondria of the null mutants contain ten times more iron than wild-type. The neurodegeneration observed in Friedreich's ataxia can be well explained on the basis of a mitochondrial iron overload responsible for an increased production of highly toxic free radicals. Rationale: Since iron accumulation is implicated in the etiology of Friedreich's ataxia, both the initial appearance of symptoms and the progression of this disease will be slowed or halted by sequestering the free iron. Transfection of ferritin-H-subfamily peptides or expression clones and/or treatment with agents that would up-regulate expression of the endogenous ferritin-H-subfamily genes will be ameliorative.
Background for atherosclerosis:
Strong epidemiological evidence is available that iron (i.e., iron excess) is an important factor in the process of atherosclerosis and that iron depletion has cardiovascular benefits and protects against ischemic heart disease. Iron-catalyzed generation of free radicals may contribute to vessel wall damage, to plaque formation and, by both mechanisms, to cardiac vessel damage. Once again, intracellular iron release from L-ferritin is implicated as a source of the iron contributing to this etiology; H-ferritin may be protective by chelating and sequestering the free and released iron. Rationale: Transfecting the appropriate cell with an ferritin-H-subfamily peptide or gene expression clone or with a gene regulator that will activate the endogenous ferritin-H-subfamily gene(s) in artery wall cells or cellular elements of athersclerotic plaques will prevent or reverse artery blockage.
Background regarding possible delivery systems and cell-targeting mechanisms:
For delivery of proteins or peptides into living cells ex vivo there are several approaches. Small peptides (about 20 kDa or smaller) may be taken up by cells without a specialized delivery system. Larger proteins may be delivered encapsulated into liposomes, liposomal constructs, or within a membrane such as a red cell ghost, and the vesicles are then made to fuse with the recipient cells by chemical means (e.g., polyethylene glycol [PEG] or calcium ions). Larger protein complexes may also be delivered encapsulated, by fusing the membranes of the capsule to the plasma membranes of the target cell. The inventors have had a large amount of experience with this type of delivery in the inventors' laboratory (references listed below). For in vivo delivery of proteins or peptides targeted to a specific cell type, the method of choice is likely to be a liposomal-type of delivery with an antibody or ligand directed at a specific cell surface protein or receptor incorporated into the liposome and the peptide or protein encapsulated within the liposome. Alternatively, a fusion protein comprised of the desired peptide (e.g., ferritin-H) fused with a protein ligand specific for the target cell receptor might be injected directly. An example of a protein ligand one might use to target hematopoietic stem cells is Stem Cell Factor (c-kit ligand) which binds to a receptor (c-kit) enriched on the surface of hematopoietic stem cells in the bone marrow. Those skilled in the art will recognize that there are a wide variety of pharmaceutical delivery mechanisms suitable for introducing proteins, protein fragments and genetic material into a cell.
For delivery of expression clones of genes encoding ferritin-H-subfamily peptides (for example), a number of plasmid carriers and transfection reagent systems are available to transfect cells ex vivo, to generate either stable transformants or transiently transfected cells, for reinfusion into the host animal or patient. Good expression plasmids are commercially available as are transfection reagents, many of the latter being cationic liposomes of one type or another. For in vivo as well as ex vivo gene transfer—that is, gene therapy—the vectors available include retroviral vectors (good only for dividing cells), adenoviral vectors (transfect many cell types, with very little cell specificity), adeno-associated viral vectors, lentiviral vectors, and electroporation systems. Any of these might be used in an ex vivo protocol where the target cells are obtained as a pure or highly enriched population, to be reinfused after gene transfer. For in vivo gene transfer, the choices are currently limited because of the difficulty of efficiently targeting specific cells with sufficient gene copies. A targeted liposome as described in the preceding paragraph is a possibility if a ligand for a high-affinity, plentiful but cell-specific receptor is incorporated.
Background regarding induction of ferritin-H gene expression in human cells:
Ferritin-H is among a group of genes that have been identified as being expressed during embryogeneis. The first major site of ferritin-H expression is in the embryonic red blood cell which is formed in the mammalian yolk sac before the blood circulation is established. This cell-specific expression of ferritin-H in early development corresponds to red cell's role as the iron storage site of the embryo. Adult red cells express much less ferritin, and iron is stored primarily in the liver (in hepatocytes) in adults where the primary ferritin expressed is ferritin-L. “Knocking out” the ferritin-H gene in mice results in intrauterine death between days 3.5 and 9.5 of development. Thus, ferritin-H is a developmentally regulated gene, expression of which is also somewhat restricted to certain cell and tissue types. The inventors have discovered that expression of ferritin-H in differentiating adult erythroid cells will reverse developmental hemoglobin switching, directly by repressing the adult β-globin gene, and either directly or indirectly causing an activation of the fetal gamma-globin gene. To activate endogenous ferritin-H gene expression in adult erythroid cells also reverses a developmental gene switch in this one cell lineage. Accomplishing this switch will, in turn, reverse another developmental switch, the hemoglobin switch, which has therapeutic benefits to people with sickle cell disease, β-thalassemia and other hemoglobinopathies. Activatinh ferritin-H expression in other cell types alleviates and protects against cancers, atherosclerosis, and neurodegenerative diseases.
It should be noted that although much is known about regulation of ferritin expression at the level of translation by iron as sensed by the IRE-binding proteins ( e.g., cytosolic cis-aconitase which is IRP-1 [IRE-binding protein-1]), this level and type of regulation does not distinguish between ferritin types. To specifically up-regulate ferritin-H expression requires regulation of the specific gene at the level of transcription.
BT-20 breast cancer cells rapidly increase ferritin-H mRNA synthesis when exposed to exogenously added heme but only slightly increase ferritin-L mRNA. This change is protective against the free radical damage of carcinogenesis. In colon cancer Caco-2 cells, ferritin-H expression leads to increased cell differentiation and a decline in the cancer phenotype. Increased ferritin-H expression also occurs during cell differentiation of erythroleukemia (K562) and hepatoma (HepG2) cell lines in culture. Although some of the DNA elements in the ferritin-H gene promoter and nuclear proteins that bind to them (e.g., P/CAF-CBP, Bbf, and NF-E2) are known, the mechanism of activation of ferritin-H transcription is not understood sufficiently to be applied clinically.
Among the exogenous factors that can be delivered/applied to cells to activate endogenous ferritin-H gene expression are heme, the phytohormone abscisic acid, and combinations of infrared and ultraviolet light, especially as applied to human keratinocytes and skin cancers.